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Biomedical subjects

M M Burger

Publications and source records attributed to M M Burger.

At least 91 records · Page 5Linked to original sources
PubMed1988

Association of polyoma virus middle T antigen and pp60src with cytoskeletal elements.

Polyoma virus middle T antigen (mT) as well as pp60v-src, the oncogene product of Rous sarcoma virus, associate with membranes and cytoskeletal structures in virus-infected cells. Furthermore, mT has been shown to be tightly bound to pp60c-src, the cellular homolog of pp60v-src. While the presence of pp60v-src in focal contacts has been demonstrated, the localization of mT and pp60c-src is less clear. We show here that mT is associated with focal contacts and that the phosphorylated protein can be immunoprecipitated with anti-vinculin serum. We suggest that the mT/pp60c-src complex and pp60v-src are tightly bound to vinculin. This interaction may be relevant in the process of cell transformation since disorganization of the actin filament network seems to play an important role in the deregulation of cell growth observed in tumor cells.

Animals↗
PubMed1987

Involvement of carbohydrates as multiple low affinity interaction sites in the self-association of the aggregation factor from the marine sponge Microciona prolifera.

Cell aggregation in the marine sponge Microciona prolifera is mediated by a multimillion molecular weight aggregation factor (MAF) and is based on two functional properties, a Ca2+-independent cell binding activity and a Ca2+-dependent factor-factor self-interaction. Monoclonal antibodies were prepared against purified MAF, and one clone was characterized which selectively inhibited the MAF-MAF association activity. Binding of the blocking antibody (Block 1) to MAF demonstrated that this epitope structure was present in 1100 copies per one MAF molecule of Mr = 2 X 10(7). Such blocking antibodies precipitated a small molecular weight protein-free glycan fraction prepared from MAF by Pronase digestion, thus indicating that the highly repetitive epitope is located in the carbohydrate portion of the molecule. Since the inhibitory activity of the Block 1 antibody could only be achieved when most of the sites were occupied by Fab fragments of this antibody, the self-association of MAF seemed to be based on the polyvalency of the carbohydrate determinants. The affinity of the protein-free glycans was very low as shown by the fact that they did not display any measurable self or MAT binding activity in their monomeric form. After cross-linking them with glutaraldehyde into polymers of the size of MAF, however, the self-interaction could be reconstituted. Thus, the MAF-MAF association activity, which is needed for cell aggregation in sponges, seems to be based on multiple low affinity carbohydrate-carbohydrate interactions, which is different from most interactions mediated by adhesion molecules characterized so far.

Amino Acids↗
PubMed1987

The cytoskeletal protein vinculin contains transformation-sensitive, covalently bound lipid.

Vinculin, which is associated with the cytoskeleton of many cells, has been suggested as a possible linker between microfilament bundles and the plasma membrane. Here it will be shown that fatty acid is covalently attached to vinculin in vivo. Furthermore, in chicken embryo fibroblasts infected with a temperature-sensitive mutant of Rous sarcoma virus, tsNY68, the acylation of vinculin at the permissive temperature was less than one-third that at the nonpermissive temperature. Thus, the covalent binding of lipid to vinculin is a transformation-sensitive event. The covalent modification of vinculin by lipids could be directly or indirectly involved in its reversible association with membranes. This modification may also provide a mechanism to alter the organization of vinculin within cells and thereby play a regulatory role in anchoring or stabilizing microfilament bundles at plasma membranes.

Actin Cytoskeleton↗
PubMed1987

Laminin is structurally conserved in the sea urchin basal lamina.

The extracellular matrix is involved in the regulation of differentiation and morphogenesis. Here we report the identification of a sea urchin embryonic extracellular matrix protein by means of a monoclonal antibody BL1 (Mab BL1) and the isolation of the protein from basal lamina preparations. In paraffin sections of fixed embryos, the antibody can be detected on the basal surfaces of cells after the blastula stage. Immunoprecipitation from embryo lysates and salt extracts of metabolically labeled basal lamina preparations demonstrates that the basal lamina antigen is a large mol. wt protein of approximate mol. wt 10 which consists of disulfide-linked subunits of mol. wts 480 000 and 260 000. Electron microscopic images show that the Mab BL1 basal lamina antigen is structurally related to the vertebrate extracellular matrix protein laminin.

Journal Article↗
PubMed1987

Detergents inhibit exocytosis in PC 12 cells: evidence for an effect on ion fluxes.

Membrane events in exocytosis were studied by examining the effect of different detergents on the K+-stimulated release of noradrenaline in the secretory cell line PC 12. The nonionic detergent Triton X-100 and the cationic detergent cetyltrimethylammonium bromide (CTAB) inhibit the noradrenaline release evoked by 55 mM K+ by 50% at very low concentrations (30 microM and 10 microM, respectively). These values are tenfold lower than the critical micellar concentrations (CMC). No such effect was seen with the anionic detergent sodium dodecyl sulphate (NaDodSO4). The inhibitory effect of 30 microM Triton X-100 is reversible, and the recovery from inhibition correlates with the loss of detergent from the cells as demonstrated by binding studies using [3H]Triton X-100. The possible relationship between this inhibition of secretion and the structural properties of the detergent was investigated. The inhibition in the presence of purified Triton X-100 subfractions turned out to be a function of the length of the oligometric ethyleneglycol chain (C6 to C26). The maximal effect was observed for Triton X-100 molecules having a chain length of 16 carbon atoms, which can penetrate just half of the lipid bilayer of the membrane. Additionally, the phase transition at 13-14 degrees C observed in an Arrhenius plot of noradrenaline release in stimulated cells was abolished. In the presence of 30 microM Triton X-100, 22Na+ uptake, 86Rb+ release, and 45Ca2+ uptake were reduced by 50-60%. These data suggest that the site of action of Triton X-100 is at the level of altering the movement of ions in PC 12 cells during the stimulatory phase of secretion.

Adenosine Triphosphate↗
PubMed1987

Docking of chromaffin granules--a necessary step in exocytosis?

Putative docking of secretory vesicles comprising recognition of and attachment to future fusion sites in the plasma membrane has been investigated in chromaffin cells of the bovine adrenal medulla and in rat phaeochromocytoma (PC 12) cells. Upon permeabilization with digitonin, secretion can be stimulated in both cell types by increasing the free Ca2+-concentration to microM levels. Secretory activity can be elicited up to 1 hr after starting permeabilization and despite the loss of soluble cytoplasmic components indicating a stable attachment of granules to the plasma membrane awaiting the trigger for fusion. Docked granules can be observed in the electron microscope in permeabilized PC 12 cells which contain a large proportion of their granules aligned underneath the plasma membrane. The population of putatively docked granules in chromaffin cells cannot be as readily discerned due to the dispersal of granules throughout the cytoplasm. Further experiments comparing PC 12 and chromaffin cells suggest that active docking but not transport of granules can still be performed by permeabilized cells in the presence of Ca2+: a short (2 min) pulse of Ca2+ in PC 12 cells leads to the secretion of almost all releasable hormone over a 15 min observation period whereas, in chromaffin cells, with only a small proportion of granules docked, withdrawal of Ca2+ leads to an immediate halt in secretion. Transport of chromaffin granules from the Golgi to the plasma membrane docking sites seems to depend on a mechanism sensitive to permeabilization. This is shown by the difference in the amount of hormone released from the two permeabilized cell types, reflecting the contrast in the proportion of granules docked to the plasma membrane in PC 12 or chromaffin cells. Neither docking nor the docked state are influenced by cytochalasin B or colchicine. The permeabilized cell system is a valuable technique for the in vitro study of interaction between secretory vesicles and their target membrane.

Adrenal Gland Neoplasms↗
PubMed1987

Digitonin-permeabilized cells are exocytosis competent.

Release of norepinephrine from PC12 cells can be stimulated by free Ca2+ in micromolar concentrations after permeabilization with 10 micrograms/ml of digitonin. This release is time and temperature dependent, half-maximal at 0.3 microM Ca2+, and, after washing out of endogenous ATP, half-maximal at about 0.5 mM MgATP when exogenously added. Similar results were obtained with bovine adrenal chromaffin cells using the same protocol. Support for the idea that the mechanism of release from both permeabilized cell types is still exocytosis is demonstrated at the electron microscopic level by immunolabeling chromaffin granule membrane antigens that were introduced into the plasma membrane following stimulation. Electron micrographs furthermore demonstrate that chromaffin granules retain typical dense cores after permeabilization, indicating that leakiness of catecholamines from the granules was not a major factor. Pores, formed by digitonin in the plasma membranes, were utilized to introduce antibodies into such exocytosis-competent cells. Anti-actin and anti-chromaffin granule membrane antibodies show a staining pattern similar to conventionally fixed and stained preparations. Our results demonstrate that pores formed by digitonin do not impair the process of exocytosis although they are big enough to allow macromolecules to pass in both directions. The digitonin-permeabilized cell is therefore an ideal in vitro system with which to study the fusion process between chromaffin granules and the plasma membrane.

Adenosine Triphosphate↗
PubMed1986

Interaction of the cytoskeletal component vinculin with bilayer structures analyzed with a photoactivatable phospholipid.

The cytoskeletal component vinculin has been proposed to act as an actin-plasma membrane linker. In order to demonstrate a possible direct interaction of vinculin with bilayers, photolabeling with a phospholipid generating a highly reactive carbene was used. This phosphatidylcholine analogue (1-palmitoyl-2-[10-[4-[(trifluoromethyl)diazirinyl]phenyl]-[3H] 9-oxaundecanoyl]-sn-glycero-3-phosphocholine), with the photoactivatable diazirine group on its apolar portion, has been shown to label selectively membrane-embedded domains of membrane proteins. Vinculin is significantly labeled upon incubation and photolysis with liposomes containing trace amounts of this photoactivatable phospholipid, but only when the liposomes also contain acidic phospholipids. Labeling of vinculin is markedly increased (5-17-fold) by all acidic phospholipids tested so far (30%, w/w), compared to labeling in neutral phospholipids. Labeling is high at low ionic strength, but significant vinculin labeling can still be observed at physiological salt concentrations and acidic phospholipid content of the membrane. Our results provide evidence that vinculin inserts into the hydrophobic part of the bilayer by interacting with acidic phospholipids. A similar interaction may be of importance in vivo.

Actins↗
PubMed1986

Reconstitution of high cell binding affinity of a marine sponge aggregation factor by cross-linking of small low affinity fragments into a large polyvalent polymer.

Species-specific reaggregation of cells from the marine sponge Microciona prolifera is mediated by a proteoglycan-like aggregation factor (MAF) of Mr = 2 X 10(7) which has two functional domains, a cell binding domain and an aggregation factor interaction domain. After extensive trypsin digestion, over 60% of the MAF mass was converted into a glycopeptide fragment of Mr = 10,000 (T-10) which is therefore a representative part of the major portion, but not of the entire MAF molecule. The T-10 fragment has a similar amino acid and carbohydrate composition as the intact MAF and displays species-specific binding. Although T-10 also inhibited MAF association with homotypic cells, its apparent affinity is 3 X 10(6) M-1, i.e. 13,000 times lower than that of native MAF. Reconstitution of binding affinity in the same order of magnitude as native MAF (Ka = 10(10) M-1) was obtained by cross-linking the glycopeptide fragment into polymers of the approximate size of MAF (Mr greater than 1.5 X 10(7) using diepoxybutane and glutaraldehyde, or periodate oxidation and glutaraldehyde. The apparent association constants of intermediate polymers with Mr = 1 X 10(5), 6 X 10(5), 9 X 10(5), 2 X 10(6) and above 1.5 X 10(7) increased proportionally to their size and were in line with association constants of MAF degradation fragments. Since the binding affinity of the T-10 glycopeptide fragment could be reconstituted by cross-linking, and since this fragment accounts for over 60% of MAF, we propose that the specificity and high affinity of the MAF-cell association is based on a highly polyvalent interaction of low affinity cell-binding sites. Such a polyvalency of the cell binding domain is advantageous for efficient cell-cell interactions and thus differs from most known interaction molecules and receptors characterized.

Amino Acids↗
PubMed1986

Structure-activity relationship in vinculin: an IR/attenuated total reflection spectroscopic and film balance study.

Surfacing and membrane-penetrating ability of vinculin and bovine serum albumin have been studied on a macroscopic level by means of a Langmuir film balance and on a molecular level by means of infrared attenuated total reflection spectroscopy. It is suggested that the driving force of the nonspontaneous process of membrane penetration by native vinculin is the spontaneous formation of rigid vinculin monolayers in the membrane. Lateral adhesion of vinculin molecules results from the formation of intermolecular pleated-sheet structures. Vinculin surface activity was found to result from an alpha-helical segment oriented approximately perpendicular to plane of the membrane. There is a conformational equilibrium between this helix and random structure. High ionic strength (110 mM) favors helix formation that leads to the greater than 100-fold enhancement of surfacing velocity relative to the velocity observed at a lower ionic strength (10 mM). Vinculin has a second helical segment oriented parallel to the plane of the membrane that is in a conformational equilibrium with the pleated-sheet structure.

Amides↗
PubMed1985

Selection of B16 melanoma cells with increased metastatic potential and low intercellular cohesion using Nuclepore filters.

A method was developed to isolate tumor cell variants which penetrate filter pores 10 X smaller than their cell diameter. Variants with very high penetration ability in a standard filter assay showed increased metastatic capacity compared to non-selected cells when assayed in vivo with intramuscular injection and removal of the primary tumor, or in a subcutaneous injection assay with the primary tumor left intact. The parent cell line, as well as randomly chosen clones displayed less metastatic capacity, which correlated with correspondingly reduced filter penetration rates. A characterization of the cells so far analyzed in vitro showed that selection through Nuclepore filters led to increased invasive spread, reduced intercellular cohesion and a somewhat higher motility. The B16 melanoma cells tested so far which showed this in vitro behavior had increased metastatic capacity, suggesting that such traits may be involved in metastatic behavior of the B16 melanoma. The procedure described for the isolation of variant tumor strains should provide cells which will help to analyze penetration phenomena in metastasis.

Animals↗
PubMed1983

Release of cell-associated concanavalin A by methyl alpha-D-mannopyranoside reveals three binding states of concanavalin-A receptors on mouse fibroblasts.

Based on the partial reversibility of concanavalin A binding by saturating concentrations of methyl alpha-D-mannopyranoside (MeManp) three states of cellular association could be characterized: type I, most rapidly established and most tightly bound, not released by MeManp at 0 degrees C or at 37 degrees C; type II, most loosely bound, released by MeManp at 0 degrees C, therefore not critically dependent on the temperature at which the release reaction is performed; type III, intermediate strength of binding, released by MeManp only at 37 degrees C, thus reflecting the temperature-sensitive nature of these cell complexes. A similar temperature dependence was found for cell-bound concanavalin A when it was displaced by an excess of the same lectin. The types of binding are seen irrespective of the temperature at which the cellular association was established. About 10% of the concanavalin A molecules bind to receptor structures in a saturable way and represent the type I association. Type I association is clearly distinct from types II and III. Type II seems to be the precursor of type III. Most of the type II associations can be converted into type III associations since ConA-cell complexes originally sensitive to the action of MeManp at low temperature gradually do become resistant after prolonged association at the same temperature. The temperature dependence in binding to cells was, however, not related to receptor mobility since glutaraldehyde-treated cells had most of the release properties that were observed in untreated cells and which did not markedly differ between 3T3 cells and their SV40-transformed counterparts. In contrast to cellular binding, dissociation of concanavalin A from Sephadex beads by saturating concentrations of MeManp was complete irrespective of temperature.

Animals↗
PubMed1983

Lectin-resistant variants of mouse melanoma cells. II. In vitro characteristics.

Lectin-resistant variants of B-16 melanoma cells were selected with wheat-germ agglutinin, ricin and concanavalin A. They exhibited altered metastasizing capacity and tumorigenicity in C57BL mice. Several in vitro properties were defined and compared including homotypic adhesiveness, microfilament organization, melanin release, activity of tyrosine hydroxylase, DNA content, and karyotypes. The possible relevance of these properties in vitro for the malignant behavior displayed in vivo is discussed. The usefulness of this approach of selecting surface variants to study the problem of metastasis is also discussed.

Animals↗
PubMed1983

Lectin-resistant B16 melanoma cells exhibit an altered response to MSH and cholera toxin.

Mouse B16 melanoma cells respond to melanocyte-stimulating hormone (MSH) or cholera toxin (CT) with an accumulation of cAMP. The kinetics and dose-response of MSH were examined in the B16 parent line and two cell clones derived from it that exhibited wheat germ agglutinin (WGA) resistance [1]. These WGA lectin-resistant cells, designated W4 and W5 showed a greater response to MSH and CT than the parent B16 cells. Exposure of the W4 and W5 cells to lotus lectin or ricin respectively, led to the previously described [2] selection of cell clones that were resistant to lotus lectin (W4L) and ricin (W5R). The W4L and W5R cells which were shown [2] to be as sensitive as the B16 parent to WGA (i.e., were phenotypically reverted to WGA sensitivity), were also found to respond to MSH in a manner similar to the B16 parent. Since lectin sensitivity has been directly correlated in these cell clones with the membrane's oligosaccharides and glycopeptide pattern, these data suggest that the cellular binding and/or biological response to hormones is influenced by the carbohydrate composition of the plasma membrane.

Animals↗